twoisoformsofaquaporin2responsivetohypertonicstressinthe bottlenose dolphin · establishment of a...

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RESEARCH ARTICLE Two isoforms of aquaporin 2 responsive to hypertonic stress in the bottlenose dolphin Miwa Suzuki 1, *, Hitomi Wakui 1 , Takuya Itou 2 , Takao Segawa 2 , Yasuo Inoshima 3 , Ken Maeda 4 and Kiyoshi Kikuchi 5 ABSTRACT This study investigated the expression of aquaporin 2 (AQP2) and its newly found alternatively spliced isoform (alternative AQP2) and the functions of these AQP2 isoforms in the cellular hyperosmotic tolerance in the bottlenose dolphin, Tursiops truncatus. mRNA sequencing revealed that alternative AQP2 lacks the fourth exon and instead has a longer third exon that includes a part of the original third intron. The portion of the third intron, now part of the coding region of alternative AQP2, is highly conserved among many species of the order Cetacea but not among terrestrial mammals. Semi- quantitative PCR revealed that AQP2 was expressed only in the kidney, similar to terrestrial mammals. In contrast, alternative AQP2 was expressed in all organs examined, with strong expression in the kidney. In cultured renal cells, expression of both AQP2 isoforms was upregulated by the addition to the medium of NaCl but not by the addition of mannitol, indicating that the expression of both isoforms is induced by hypersalinity. Treatment with small interfering RNA for both isoforms resulted in a decrease in cell viability in hypertonic medium (500 mOsm kg -1 ) when compared with controls. These findings indicate that the expression of alternatively spliced AQP2 is ubiquitous in cetacean species, and it may be one of the molecules important for cellular osmotic tolerance throughout the body. KEY WORDS: Cellular osmoregulation, Osmotic tolerance, Alternative splicing, Cetacea INTRODUCTION Cetaceans, i.e. whales and dolphins, are adapted to an aquatic life in seawater. The skin and digestive tracts are unavoidably involved in confronting osmotic stress. Plasma osmolality of cetaceans, approximately 310360 mOsm kg -1 , is up to 75 mOsm kg -1 higher than that in terrestrial mammals, and their plasma Na + concentration would be indicative of dehydration in humans (Ortiz, 2001). In addition, their plasma osmolality fluctuates widely; for example, bottlenose dolphins can be tolerant against fluctuation in osmotic stress levels as their plasma osmolality is in the range of 319 to 358 mOsm kg -1 (Ridgway and Venn-Watson, 2010), in contrast to terrestrial mammals, in which internal fluid osmolality is rigidly controlled in a narrow range to prevent cellular damage. What are the osmoregulatory mechanisms that can facilitate resistance to such elevations and fluctuations in the osmotic and electrolyte content of extracellular fluid in cetaceans? Studies on this question have been non-existent so far. Aquaporins (AQPs) are water channel proteins that confer the selective plasma membrane water permeability required for rapid and regulated physiological processes, such as secretion and reabsorption (Gomes et al., 2009). In terrestrial mammals, AQP2 expression is limited to the principal cells of the renal collecting duct, where it functions in the reabsorption of water from primitive urine by trafficking through cells from the endosomal compartments to the apical membrane under the control of vasopressin (Coleman et al., 2000; Fushimi et al., 1997; Nielsen et al., 1999). In the principal cells of the renal inner medulla, AQP2 expression is induced by hypertonicity (Hasler, 2009; Kasono et al., 2005; Storm et al., 2003). Osmotic tolerance is important for maintaining cellular shape in eukaryotes and is crucial in renal cells, which are confronted with high fluctuations in intra- and extracellular concentrations of urea and NaCl during antidiuresis and diuresis (Beck et al., 1998). Cells defend against severe osmotic stress by dramatically increasing and decreasing intracellular organic osmolyte concentrations (Neuhofer and Beck, 2006). In response, water molecules passively flow according to osmotic gradients to balance the osomolality between intra- and extracellular fluids. Under hypertonic conditions, tonicity-responsive enhancer binding protein (TonEBP/NFAT5) binds to the tonicity-responsive enhancer cis element located upstream of AQP2, upregulating AQP2 expression (Miyakawa et al., 1999). Increased AQP2 expression increases water flux across the plasma membrane, thereby increasing the water permeability of principal cells. For these characteristics of AQP2 and in view of reports that AQP2 was positively selected during the secondary adaptation of cetacean species to the marine environment (Nery et al., 2013; Xu et al., 2013; Yim et al., 2014), for this study it was hypothesized that AQP2 could play some roles in cellular osmoregulation in cetaceans, who are living in a hypertonic environment in comparison to their body fluids. It was previously reported that AQP2 is distributed in the principal cells of the kidneys of the bottlenose dolphin and Bairds beaked whale, similar to terrestrial mammals (Suzuki et al., 2008). During the process of the present study, unique data were unexpectedly obtained from rapid amplification of cDNA ends (RACE)-PCR experiments, indicating alternative splicing of the AQP2 gene expressed in many organs of bottlenose dolphin. Many genes undergo alternative splicing, resulting in proteome complexity by providing different protein isoforms from a single gene, and it sometimes provides a spark for evolution (Mironov et al., 1999; Modrek and Lee, 2002, 2003; Tarrío et al., 2008; Woodley and Valcarcel, 2002). Thus, alternative splicing of AQPs Received 30 September 2015; Accepted 15 February 2016 1 Department of Marine Science and Resources, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan. 2 Nihon University Veterinary Research Center, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan. 3 Cooperative Department of Veterinary Medicine, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan. 4 Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan. 5 Fisheries Laboratory, The University of Tokyo, 2941-4 Bentenjima, Maisaka, Nishi, Hamamatsu, Shizuoka 431-0214, Japan. *Author for correspondence ([email protected]) 1249 © 2016. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2016) 219, 1249-1258 doi:10.1242/jeb.132811 Journal of Experimental Biology

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Page 1: Twoisoformsofaquaporin2responsivetohypertonicstressinthe bottlenose dolphin · establishment of a dolphin renal cell line, the kidney of a female bottlenose dolphin that had been

RESEARCH ARTICLE

Two isoforms of aquaporin 2 responsive to hypertonic stress in thebottlenose dolphinMiwa Suzuki1,*, Hitomi Wakui1, Takuya Itou2, Takao Segawa2, Yasuo Inoshima3, Ken Maeda4 andKiyoshi Kikuchi5

ABSTRACTThis study investigated the expression of aquaporin 2 (AQP2) and itsnewly found alternatively spliced isoform (alternative AQP2) and thefunctions of these AQP2 isoforms in the cellular hyperosmotictolerance in the bottlenose dolphin, Tursiops truncatus. mRNAsequencing revealed that alternative AQP2 lacks the fourth exonand instead has a longer third exon that includes a part of the originalthird intron. The portion of the third intron, now part of the codingregion of alternative AQP2, is highly conserved among many speciesof the order Cetacea but not among terrestrial mammals. Semi-quantitative PCR revealed that AQP2 was expressed only in thekidney, similar to terrestrial mammals. In contrast, alternative AQP2was expressed in all organs examined, with strong expression inthe kidney. In cultured renal cells, expression of both AQP2isoforms was upregulated by the addition to the medium of NaClbut not by the addition of mannitol, indicating that the expression ofboth isoforms is induced by hypersalinity. Treatment with smallinterfering RNA for both isoforms resulted in a decrease in cellviability in hypertonic medium (500 mOsm kg−1) when compared withcontrols. These findings indicate that the expression of alternativelyspliced AQP2 is ubiquitous in cetacean species, and it may be oneof the molecules important for cellular osmotic tolerance throughoutthe body.

KEY WORDS: Cellular osmoregulation, Osmotic tolerance,Alternative splicing, Cetacea

INTRODUCTIONCetaceans, i.e. whales and dolphins, are adapted to an aquatic lifein seawater. The skin and digestive tracts are unavoidably involvedin confronting osmotic stress. Plasma osmolality of cetaceans,approximately 310–360 mOsm kg−1, is up to 75 mOsm kg−1

higher than that in terrestrial mammals, and their plasma Na+

concentration would be indicative of dehydration in humans (Ortiz,2001). In addition, their plasma osmolality fluctuates widely; forexample, bottlenose dolphins can be tolerant against fluctuation inosmotic stress levels as their plasma osmolality is in the range of 319to 358 mOsm kg−1 (Ridgway and Venn-Watson, 2010), in contrastto terrestrial mammals, in which internal fluid osmolality is rigidly

controlled in a narrow range to prevent cellular damage. What arethe osmoregulatory mechanisms that can facilitate resistance to suchelevations and fluctuations in the osmotic and electrolyte content ofextracellular fluid in cetaceans? Studies on this question have beennon-existent so far.

Aquaporins (AQPs) are water channel proteins that confer theselective plasma membrane water permeability required for rapidand regulated physiological processes, such as secretion andreabsorption (Gomes et al., 2009). In terrestrial mammals, AQP2expression is limited to the principal cells of the renal collectingduct, where it functions in the reabsorption of water from primitiveurine by trafficking through cells from the endosomal compartmentsto the apical membrane under the control of vasopressin (Colemanet al., 2000; Fushimi et al., 1997; Nielsen et al., 1999). In theprincipal cells of the renal inner medulla, AQP2 expression isinduced by hypertonicity (Hasler, 2009; Kasono et al., 2005; Stormet al., 2003). Osmotic tolerance is important for maintaining cellularshape in eukaryotes and is crucial in renal cells, which areconfronted with high fluctuations in intra- and extracellularconcentrations of urea and NaCl during antidiuresis and diuresis(Beck et al., 1998). Cells defend against severe osmotic stress bydramatically increasing and decreasing intracellular organicosmolyte concentrations (Neuhofer and Beck, 2006). In response,water molecules passively flow according to osmotic gradients tobalance the osomolality between intra- and extracellular fluids.Under hypertonic conditions, tonicity-responsive enhancer bindingprotein (TonEBP/NFAT5) binds to the tonicity-responsive enhancercis element located upstream of AQP2, upregulating AQP2expression (Miyakawa et al., 1999). Increased AQP2 expressionincreases water flux across the plasma membrane, therebyincreasing the water permeability of principal cells.

For these characteristics of AQP2 and in view of reports thatAQP2 was positively selected during the secondary adaptation ofcetacean species to the marine environment (Nery et al., 2013;Xu et al., 2013; Yim et al., 2014), for this study it was hypothesizedthat AQP2 could play some roles in cellular osmoregulation incetaceans, who are living in a hypertonic environment incomparison to their body fluids. It was previously reported thatAQP2 is distributed in the principal cells of the kidneys of thebottlenose dolphin and Baird’s beaked whale, similar to terrestrialmammals (Suzuki et al., 2008). During the process of the presentstudy, unique data were unexpectedly obtained from rapidamplification of cDNA ends (RACE)-PCR experiments,indicating alternative splicing of the AQP2 gene expressed inmany organs of bottlenose dolphin.

Many genes undergo alternative splicing, resulting in proteomecomplexity by providing different protein isoforms from a singlegene, and it sometimes provides a spark for evolution (Mironovet al., 1999; Modrek and Lee, 2002, 2003; Tarrío et al., 2008;Woodley and Valcarcel, 2002). Thus, alternative splicing of AQPsReceived 30 September 2015; Accepted 15 February 2016

1Department of Marine Science and Resources, College of Bioresource Sciences,Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan. 2NihonUniversity Veterinary Research Center, Nihon University, 1866 Kameino, Fujisawa,Kanagawa 252-0880, Japan. 3Cooperative Department of Veterinary Medicine,Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan. 4Joint Faculty of VeterinaryMedicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan.5Fisheries Laboratory, The University of Tokyo, 2941-4 Bentenjima, Maisaka, Nishi,Hamamatsu, Shizuoka 431-0214, Japan.

*Author for correspondence ([email protected])

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is assumed to occur as with other genes; however, only a few casesof alternative expression of AQP family proteins in mammals havebeen reported (Moe et al., 2008; Amiry-Moghaddam et al., 2005).To elucidate the biological implication of alternative splicing ofAQP2, details on the expression of AQP2 and its alternativelyspliced isoform (alternative AQP2) in the bottlenose dolphin, thefunction of the isoforms for cellular hyperosmotic/hyperosmolalitytolerance, and the universality of alternative AQP2 expression incetaceans were investigated in this study.

MATERIALS AND METHODSAll experiments were conducted according to the FundamentalGuidelines for Proper Conduct of Animal Experiment and RelatedActivities in Academic Research Institutions under the jurisdictionof the Ministry of Education, Culture, Sports, Science andTechnology and the guidelines for animal experiments in theCollege of Bioresource Sciences, Nihon University.

Samples and tissue preparationIn January 2010, 19 organs were obtained from a male and a femalebottlenose dolphin, Tursiops truncatus (Montagu 1821) (sampleIDs: 10TH009 and 10TH014), under the cooperation of thescientific surveys by the National Research Institute of Far SeasFisheries, Fisheries Research Agency of Japan. The organs werecollected immediately post mortem. Samples were taken from eachorgan, and sub-samples were preserved in RNAlater (Ambion/LifeTechnologies, Carlsbad, CA, USA) at −20°C for RNA isolationand semi-quantitative analysis. Other sub-samples were frozen inliquid nitrogen to extract proteins for immunoblotting. Forimmunohistochemistry, tissues were cut into small blocks andfixed in 4% paraformaldehyde (Wako, Osaka, Japan). For theestablishment of a dolphin renal cell line, the kidney of a femalebottlenose dolphin that had been kept clinically healthy for morethan 36 years but died in February 2013 at Izu Mito Sea Paradise,Shizuoka, Japan, was used. For genome analyses, tissues of sixother cetacean species in five families were collected. Details oneach specimen of the six species are shown in Table 1.

Isolation of full-length cDNA of the new aquaporinFull-length cDNAs encoding the AQP2 isoforms were isolatedusing the method of Suzuki (2010). Briefly, total RNAwas isolatedfrom the kidney of the bottlenose dolphins using Isogen reagents(NipponGene, Chiyoda, Tokyo, Japan). After DNA degradation bydeoxyribonuclease treatment, 5 μg of RNAwas reverse transcribedto cDNA using the GeneRacer® kit for RLM-RACE (Invitrogen/Life Technologies). The AQP2 cDNA coding region was first

amplified with primers (AQP2, FW1 and RV1; Table S1) usingstandard procedures to design gene-specific primers. 5′ and 3′RACE PCR were carried out using the primers (each of AQP2-GRRV and alternative AQP2-GRRV was used with the 5′ primerfor 5′ RACE; each of AQP2-GRFW3, AQP2-GRFW4, alternativeAQP2-GRFW1 and alternative AQP2-GRFW2was used with the 3′primer for 3′ RACE; Table S1) according to the manufacturer’sinstructions. PCR products were size-fractionated, purified andsubcloned into the pGEM T-Easy Vector System (Promega,Madison, WI, USA) followed by the TA cloning, and thenucleotide sequences were determined using a DNA sequencer.Transmembrane sites of alternative AQP2 were predicted usingTMPred (http://ch.embnet.org/software?TMPRED_form.html).

Preparation of constructsEach region containing the full open reading frame (ORF) ofdolphin AQP2 and alternative AQP2 was amplified by reversetranscription PCR (RT-PCR) using kidney cDNA and specificprimers (AQP2-FW2 and -RV2 for AQP2, AQP2-FW2 andalternative AQP2-RV2 for alternative AQP2; Table S1). The PCRproducts were each cloned into the pGEM T-Easy Vector system(Promega), and the plasmids were purified via transformation inJM109 (NipponGene). Fragments containing the ORF were cutfrom the plasmid using EcoRI (Takara, Kusatsu, Shiga, Japan) andsubsequently ligated into the 3Z vector (Promega) linearized by thesame enzyme. Using the plasmid obtained, capped AQP2 andalternative AQP2 cRNAs with a poly-A tail were transcribed in vitrousing the mMessage mMachine SP6 kit (Ambion/LifeTechnologies) after digestion with NaeI (Takara) for AQP2 andBtsI (Takara) for alternative AQP2.

Water permeability testsWater permeability tests were performed as reported previously(Suzuki, 2010). Briefly, Xenopus laevis oocytes were defolliculatedwith collagenase I and microinjected with 10 ng of the synthesizedcRNA or 50 nl of distilled water as a control and incubated at 18°Cfor 48 h in modified Barth’s saline (MBS). After incubation,oocytes were transferred from 200 mOsm kg−1 H2O to70 mOsm kg−1 H2O in MBS diluted with distilled water. Aphotograph of the oocytes silhouette was taken every 20 s for upto 2 min or until the time of oocyte rupture. The oocyte volume wascalculated from its cross-sectional area. The osmotic waterpermeability (Pf ) was calculated according to Preston et al.(1992). To test sensitivities of the aquaporins to HgCl2, whichblocks the path for water molecules of aquaporins, the effects ofmercuric chloride were examined by incubating oocytes in MBScontaining 3 µmol l−1 HgCl2 for 15 min before the test. Significantdifferences in Pf values were determined by one-way ANOVA,followed by the Steel–Dwass multiple comparison method.

Determination of AQP2 and alternative AQP2 tissueexpressionTo determine the tissue expression of AQP2 and alternative AQP2,semi-quantitative RT-PCR was performed for each gene. TotalRNAwas isolated from tissue samples of 19 organs (brain, pituitary,epidermis, muscle, heart, kidney, adrenal gland, testis, ovary,placenta, forestomach, main stomach, pyloric stomach, smallintestine, large intestine, liver, pancreas, spleen and lung) of themale bottlenose dolphin. A total RNA sample (5 μg) obtained fromeach organ was reverse transcribed into cDNA using the Ready-to-Go kit (Promega). RT-PCR of AQP2 and alternative AQP2 wasconducted using the cDNAs and gene-specific primer sets (AQP2-

Table 1. Samples for genomic PCR

Species Sex Origin

Minke whale Balaenopteraacutorostrata

M Stranded in Shizuoka,Shizuoka, 4 Apr 2007

Dwarf sperm whale Kogia sima F Stranded in Honmoku,Kanagawa, 6 Nov 2008

Baird’s beaked whale Berardiusbairdii

M Stranded in Arasaki,Kanagwa, 27 Jul 2005

Pseudo killer whale Pseudorcacrassidens

M Died in Hakkeijima SeaParadise, 9 Sep 2009

Pacific white-sided dolphinLagenorhynchus obliquidens

M Stranded in Chita, Aichi,16 May 2007

Finless porpoise Neophocaenaphocaenoides

F Stranded in Hokota,Ibaraki, 6 Nov 2005

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FW1 and -RV1 for AQP2, alternative AQP2-FW and -RV1 foralternative AQP2; Table S1). Primers for amplification of eachAQP2 were constructed to include the full ORF, and those forGAPDH were described by Beineke et al. (2004). The sequences ofPCR products were confirmed via TA cloning.

Immunohistochemistry and immunoblottingImmunohistochemical localization of AQP2 and alternative AQP2in the kidney of the dolphin was determined using the methodpreviously described (Suzuki et al., 2008). Alternative AQP2distribution was also detected in another 15 organs (epidermis,esophagus, forestomach, main stomach, pyloric stomach, smallintestine, liver, pancreas, spleen, adrenal gland, ovary, lung, muscle,heart and brain). Briefly, 5 μm paraffin sections were prepared, thenafter deparaffinization, the sections were soaked in 0.6% H2O2 inmethanol for 30 min to inactivate the endogenous peroxidase. Afterwashing and blocking in 10% non-immune goat serum for 30 min,sections were incubated in a 1:1000 dilution (0.8 μg ml−1 for AQP2and 6.9 μg ml−1 for alternative AQP2) of each antibody or rabbitIgG (2 μg ml−1) overnight at 4°C. After washing, the sections wereincubated with HRP-labeled polymer anti-rabbit IgG (DakoEnVision+ System; Agilent Technologies, Santa Clara, CA, USA)for 30 min and washed with PBS. The sections exposed toantibodies were reacted with 0.02% diaminobenzidinetetrahydrochloride (DAB, Wako) in PBS with 0.02% H2O2 forcolorization. Localization of AQP2 and alternative AQP2 inXenopus oocytes injected with each cRNA was determined byfluorescent immunostaining using 1:5000 dilutions of goat anti-rabbit IgG (H+L) secondary antibody (Alexa Fluor 594 conjugate;Invitrogen/Life Technologies) instead of the HRP-labeled polymer.Immunoblots were visualized using the Vectastain Elite ABC kit

(Vector Laboratories, Burlingame, CA, USA) according to themethod reported previously (Suzuki et al., 2008). Briefly, frozendolphin kidney tissue was thawed and homogenized in buffercontaining a protease inhibitor. The homogenate was centrifuged andthe supernatant was mixed with an equal volume of 2× sample bufferand heated. The fraction was submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a 5–20% polyacrylamidegel, and separated proteinswere electrotransferred to a polyvinylidenedifluoridemembrane.Afterwashing and blocking, themembranewasincubated with 1:1000 dilutions of each antibody overnight at 4°C.Following washing, the membrane was incubated with biotinylatedanti-rabbit IgG. After washing, the blot was incubated with the ABCmixture and visualized using DAB.

AntibodiesAnti-AQP2 antibody was affinity-purified anti-rabbit antibodyagainst a synthetic peptide corresponding to the 15 carboxyl-terminal amino acid residues of rat AQP2 (Alomone Laboratories,Jerusalem, Israel). Anti-alternative AQP2 antibody was raised usingantigen elaborated from the specific amino acid sequence at theposition 204–222 (MAETSLPSPPPETHLAGR). Rabbits wereimmunized with the antigen conjugated with keyhole limpethemocyanin and serum was retrieved. Antibody was purified fromserum using protein A.

Effects of hyperosmotic environment on expression of theAQP2 isoforms in bottlenose dolphin renal cellsCell cultureDolphin primary renal cells were isolated from the kidney of afemale bottlenose dolphin that died at Izu Mito Sea Paradise.Isolated cells were maintained in RPMI 1640 (Wako) supplemented

with 15% cosmic calf serum (HyClone; Thermo Fisher Scientific,Provo, UT, USA), 1% non-essential amino acids (Gibco, LongIsland, NY, USA), 1 mmol l−1 sodium pyruvate (Gibco) andZellShield (Minerva Biolabs, Berlin, Germany). After fourpassages, an expression plasmid DNA encoding the large Tantigen origin-defective simian virus 40 (SV40) was transfectedinto the renal cells using Lipofectamine 2000 (Invitrogen/LifeTechnologies). Transfected cells were selected by addition of G418and maintained in Dulbecco’s modified Eagle’s minimal essentialmedium (DMEM) supplemented with 10% fetal calf serum,100 U ml−1 streptomycin and 100 mg ml−1 penicillin. Theestablished renal cells were designated as Dol KT1 cells.

NaCl and mannitol treatmentDol KT1 cells were cultured in 6-well plates until reachingapproximately 90% confluence. NaCl or mannitol was added tothe medium to 350 or 500 mOsm kg−1 H2O, respectively. Afterincubation for 24 h in a CO2 incubator at 37°C, cells were lysed in1 ml of Isogen (Nippon Gene).

Real-time PCRTotal cellular RNA was extracted using Isogen according to themanufacturer’s instructions. cDNA was synthesized using 1 µg oftotal RNA with the High-capacity RNA-to-cDNA kit (LifeTechnologies). Real-time PCR was performed using the cDNAmixed with 0.3 μmol l−1 primers and Quantitect SYBR GreenMix (Qiagen, KJ, Venlo, The Netherlands) using Rotor-Gene Q(Qiagen). Primer sequences used for real-time PCR of GAPDH,AQP2 and alternative AQP2 are shown in Table S1 (AQP2-FW3and -RV3 for AQP2, AQP2-FW3 and alternative AQP2-RV3 foralternative AQP2, and GAPDH-FW and -RV for GAPDH).

RT-PCR to assess tonicity-enhancer binding protein expressionTo ascertain TonEBP expression in the cells, RT-PCR wasconducted using the standard procedure with the synthesizedcDNA and specific primers to amplify its full-length ORF (TonEBPFW1-RV1; Table S1). Changes in TonEBP mRNA expression byaddition of NaCl to medium to 350 and 500 mOsm kg−1 H2O werealso tested by semi-quantitative RT-PCR using the cDNAs aboveand another set of primers (FW2-RV2; Table S1).

StatisticsSignificant differences in mRNA quantities were determined byone-way ANOVA, followed by Scheffe’s paired comparison test.

RNA interference of AQP2 and alternative AQP2 expressionEffect of knockdown in hyperosmotic mediumsiRNAs were designed to correspond to positions unique to AQP2and alternative AQP2 mRNAs, as shown in Table S2; the siRNAswere then synthesized (Hokkaido System Science Co., Sapporo,Hokkaido, Japan). Eighty percent confluent Dol-KT1 cells wereprepared in DMEM with 10% fetal bovine serum in 6-well plates,and 2.5 μg of siRNA for each of AQP2, alternative AQP2 andnegative control siRNA (Allstar Negative Control siRNA, Qiagen)were transfected into the cells using Lipofectamine 3000(Invitrogen/Life Technologies) in Opti-MEM (Invitrogen/LifeTechnologies). After 72 h incubation to complete the siRNAtransfection, NaCl was added to the medium to 500 mOsm kg−1

H2O. After 48 h of incubation in the hypertonic medium, cells werephotographed and tested for viability using the CellTiter-GloLuminescent Cell Viability Assay kit (Promega) according to themanufacturer’s instructions. Luminescence intensity of each well,

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representing ATP content, was measured by a luminometer.Numbers of cells in photographs were also counted to comparethe cell survival rate.

Knockdown efficiency testTo confirm siRNA transfection, cells were co-transfected withpsiCHECK-1 vector (Promega), ligated to the ORF of GAPDH,AQP2 and alternativeAQP2 cDNA, and 2.5 μg of each siRNA into90%confluentDol-KT1 cells.After 72 h incubation, luciferase activitywas evaluated using the Renilla Luciferase Assay System (Promega),comparing the activity with or without transfection of each siRNA.

StatisticsSignificant differences in luminescience intensities, cell numbersand luciferase activity were determined by one-way ANOVA,followed by Scheffe’s paired comparison test.

Genomic analysesTo determine whether the alternative AQP2 can be expressedin multiple species, the sequence of the region comprising thethird exon and the following intron up to the stop codon of thealternative AQP2 mRNAwas determined in seven cetacean speciesrepresenting five families (Balaenidae: Minke whale, Balaenopteraacutorostrata; Kogiidae: dwarf sperm whale, Kogia sima;Ziphiidae: Baird’s beaked whale, Berardius bairdii; Delphinidae:bottlenose dolphin, Pacific white-sided dolphin, Lagenorhynchusobliquidens, and pseudo killer whale, Pseudorca crassidens; andPhocoenidae: finless porpoise, Neophocaena phocaenoides).Tissue samples were collected post mortem from captive orstranded animals (Table 1), and genomic DNA was extractedusing the Wizard® Genomic DNA Purification Kit (Promega). PCRwas performed to amplify AQP2 using specific primers (AQP2-FW1 and AQP2-RV1; Table S1). Via TA subcloning, the regionscomprising intron 2 to exon 4 were sequenced.The sequence in the dolphin was aligned using mVISTA (Frazer

et al., 2004) for cetaceans and their closely related ungulates (cow,Bostaurus, Ensembl: ENSBTAG00000008374; bison, Bison bison,NW_011494955; chiru, Pantholops hodgsonii, NW_005816316;sheep, Ovis aries, Ensembl: ENSOARG00000018180; pig, Susscrofa, ENSSSCG00000021193; camel, Camelus bactrianus,XM_010964262). To compare the substitution rate, mVISTA wasalso run for four families in Ruminantia (cow, bison, chiru and sheep)that diverged 31.6 million years ago, almost at the same as cetaceans(32.3 million years ago), using the sequence in cow as a reference.

RESULTSFull-length cDNAs of two AQP2 isoforms were isolated fromthe kidney of the bottlenose dolphinThe structures of AQP2 and full-length AQP2 and alternative AQP2mRNA are presented in Fig. 1. The gene comprises four exons andthree introns, and comparison of the AQP2 and alternative AQP2sequences with the genomic DNA of bottlenose dolphin AQP2(ENSTTRG00000001003, GeneScaffold 1570) revealed thatalternative AQP2 has a longer third exon, where the 3′ sequencecorresponds to the third intron, and it also lacks the fourth exon. Thealternative AQP2 mRNA sequence comprises 988 bp, including a744 bp ORF encoding 247 amino acids (DDBJ accession no.LC053642); AQP2 cDNA comprises 1411 bp, including a 916 bpORF encoding 271 amino acids (DDBJ accession no. LC053641).Both of the AQP2 isoforms have two asparagine–proline–alanine(NPA) boxes (Fig. 1B). The alternative AQP2 lacks thephosphorylation sites (serine 256 and 261) present in the C terminus

ofAQP2, which are responsible for vasopressin-stimulated traffickingto the plasma membrane (Hoffert et al., 2006). Six segments, aminoacid positions 12–32, 37–57, 86–106, 130–150, 157–177 and 227–247, in alternative AQP2 were predicted as transmembrane sequencesby hydrophybicity predicted transmembrane sites (Fig. 2C).

Two AQP2 isoforms demonstrate water permeabilityThe water permeability of Xenopus oocytes injected with bottlenosedolphin AQP2 cRNA, alternative AQP2 cRNA or water (control)was determined to test the aquaporin properties. The swelling rate ofoocytes expressing AQP2 was 1.2 times that of controls (Fig. 2).The water permeability of oocytes expressing alternative AQP2 washigher than that of controls but lower than that of oocytes expressingAQP2. Treatment with 3 μmol l−1 HgCl2 significantly decreased thewater permeability of oocytes expressing both isoforms (Fig. 2A,B).The expression of each protein was confirmed by fluorescentimmunostaining (Fig. 2C). Fluorescence was concentrated on theplasma membrane in oocytes expressing AQP2, but in both theplasma membrane and intracellular compartments in thoseexpressing alternative AQP2.

AQP2 is expressed only in the kidney, but alternative AQP2 isubiquitously expressed in bottlenose dolphin organsSemi-quantitative RT-PCR was used to investigate the expression ofAQP2 and alternative AQP2 in 19 organs. As shown in Fig. 3A, thePCRproduct forAQP2 (813 bp)was observedonly in the kidney. ThePCR product for alternative AQP2 (915 bp) was present in all 19organs tested, with the most intense band appearing in the kidney.

AQP2 and alternative AQP2 were localized in a differentdistribution in the kidneyThe tissue distribution of the two AQP2 isoforms in the bottlenosedolphin kidney was examined using immunohistochemical analysisof paraffin sections (Fig. 3B). Intense positive staining was observedin the renal medulla, particularly in the papilla, for both AQP2isoforms. AQP2was detected in the apical membrane of the principalcells of the renal collecting duct. Intense positive staining foralternative AQP2 was observed in the principal cells of the duct andtransitional cells of the renal papilla; only weak stainingwas observedin other cells. Intense bands were observed at the anticipated positions(28.9 and 25.7 kDa, respectively) in immunoblotting with both anti-AQP2 and anti-alternative AQP2 antibodies. Light, smeared bandspresumed to be glycosylated forms of each protein were observedabove the intense bands (Fig. 3B).

Transcriptional activity of AQP2 and alternative AQP2 wereincreased in renal cells exposed to hypersaline mediumTo investigate mRNA expression of AQP2 and alternative AQP2 inresponse to elevated NaCl levels or increased osmolality, NaCl ormannitol was added to the medium with cultured bottlenose dolphinrenal cells. AQP2 mRNA substantially increased (478±59%;Fig. 4A) as did alternative AQP2 mRNA (481±49%) underelevated levels of NaCl (500 mOsm kg−1 medium; Fig. 4B).Alternative AQP2 mRNA expression tended to increase undermildly increased levels of NaCl (350 mOsm kg−1 medium; 315±101% of control) but without statistical significance (P=0.15).Hyperosmolality induced by the addition of mannitol did notchange the mRNA expression of either AQP2 isoform. RT-PCRwas performed to confirm TonEBP/NFAT5 expression in the renalcells, revealing amplification of the full-length 4605 bp ORF(Fig. 4C) with a sequence consistent with the registered sequence(XM_004312069). TonEBP mRNA expression was increased by

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addition of NaCl to the medium (146±12% and 277±7% in 350 and500 mOsm kg−1 H2O, respectively; Fig. 4C).

RNA interference of AQP2 and alternative AQP2 resulted incell death in hypersaline mediumTo reveal the function of each AQP2 in cellular osmotic tolerance,ATP content was measured to determine cell viability uponexposure to medium containing increased levels of NaCl for 48 hon each line of AQP2 knockdown cells. In the medium withelevated NaCl levels, ATP content was decreased to 44.4±2.1%for AQP2 knockdown cells and to 24.1±0.6% for alternativeAQP2 knockdown cells compared with that of the cells in normalmedium (Fig. 5A). In contrast, cell viabilities of siRNA-free andnegative control groups did not change significantly (93.4±0.6%and 87.9±1.4%, respectively). Numbers of knockdown cells weresignificantly decreased by NaCl addition (448±32 cells cm−2 in

control, 160±31 cells cm−2 in AQP2 siRNA, 136±13 cells cm−2 inalternative AQP2 mRNA; P<0.001). The cells transfected withAQP2 and alternative AQP2 siRNAs shrank severely when exposedto medium with increased NaCl levels (Fig. S2).

To check the siRNA knockdown efficiency, luciferase assayswere performed using cells transfected with the psiCHECK vectorligated to cDNA of GAPDH, AQP2 or alternative AQP2 with orwithout co-transfection of each siRNA (Fig. 5B). In all groups,the luminescence intensity was suppressed to very low levels bysiRNA (GAPDH, 8.4±0.7%; AQP2, 28.2±10.6%; alternativeAQP2, 13.2±3.3%) compared with controls without siRNA.

The coding region of alternative AQP2 is highly conservedonly in CetaceaThe sequence through the third exon to the stop codon of alternativeAQP2 (219 bp) in AQP2 genes of seven cetacean species was

Authentic AQP2 mRNA

Alternatively spliced AQP2 mRNA

Dolphin AQP2 gene

Start Stopaaa

ATG TGA916 bp

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ATG TGA744 bp

A

BAQP2 MWELRSIAFSRAVFAEFLATLVFVFFGLGSALNWPQALPSVLQIAMAFGLAIGTLVQALG 60Alternative AQP2 ------------------------------------------------------------ 60

AQP2 HVSGAHINPAVTVACLVGCHISFLRAAFYVAAQLLGAVAGAALLSEITPPDIRGDLAVNA 120Alternative AQP2 ------------------------------------------------------------ 120

AQP2 LSNNSTAGQAVTVELFLTLQLVLCIFASTDERRGDNLGTPALSIGFSVVLGHLLGIHYTG 180Alternative AQP2 ------------------------------------------------------------ 180

AQP2 CSMNPARSLAPAVVTGKFDNHWVFWVGPLVGAILASLLYNYILFPPAKSLSERLAVLKGL 240Alternative AQP2 -----------------------MAETSLPSPPPETHLAGRAWGGISKPSTVLLLGPPWS 240

AQP2 EPDTDWEEREVRRRQSVELHSPQSLPRGSKA 271Alternative AQP2 LGSTLQV 247

**

3126 bp 390 bp

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Fig. 1. Splicing pattern of AQP2 in thebottlenose dolphin. (A) Structures ofAQP2 gene, AQP2 mRNA andalternative AQP2 mRNA. Filled barsrepresent exons. Authentic AQP2mRNAcomprises exons 1, 2, 3 and 4 (total,1411 bp; ORF, 916 bp), and alternativeAQP2 mRNA contains exons 1, 2and x3′ (total, 988 bp; ORF, 744 bp).(B) Alignment of the deduced AQP2 andalternativeAQP2 amino acid sequences.Hyphen indicates the same residue asthat in AQP2. Underlines indicate theasparagine–proline–alanine motifshighly conserved in aquaporin familymembers. Asterisks indicate serineresidues that are phosphorylation sitesresponsible for AQP2 trafficking to theplasma membrane. (C) Kyte andDoolittle hydropathy profile of the dolphinalternative AQP2 protein. Arrowsindicates the locations of thetransmembrane domain.

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determined to check for the universality and possibility of alternativeAQP2 expression. As shown in Fig. 6A and Fig. S3, the region isquite highly conserved among the species, with neither inserts nordeletions. The sequences in the homologous region of the intron inRuminantia with a similar divergent time to Cetacea are not highlyconserved (Fig. 6A). The corresponding regions in Artiodactyla arenot similar to those of the cetacean AQP2, with several insertionsand deletions, indicating that alternative AQP2 is highly unlikely tobe expressed in the same form in those mammals (Fig. 6B).

DISCUSSIONThis study revealed the expression of two AQP2 isoforms inbottlenose dolphin. One is what might be considered to be normalAQP2 in non-cetacean species, and is expressed only in the kidneyand has high water permeability, similar to AQP2 of othermammals. The other is alternatively spliced AQP2 expressed in

multiple organs with lower Pf values. Almost all instances ofalternative splicing result from the use of one or more than four basicmodules: alternative 5′ splice-site choice, alternative 3′ splice-sitechoice, cassette-exon inclusion or skipping, or intron retention(Keren et al., 2010; Nilsen and Graveley, 2010). The dolphinalternative AQP2 reported in this study is the result of intronretention.

Analyses of AQP2 in six cetacean species, in the familiesBalaenidae, Kogiidae, Ziphiidae, Delphinidae and Phocoenidae,indicate that the same pattern of alternative AQP2 splicing isprobably present in a wide range of cetacean species. However,AQP2 in closely related artiodactyl species, such as pig, cow, sheep,camel and other terrestrial mammals, do not have the base sequencecorresponding to the alternatively spliced region in the third intronof cetaceans. Besides, the region of concern is not very wellconserved among Ruminantia, which diverged later than the orderCetacea. Thus, the newly acquired exon region is uniquelyconserved in the cetacean lineage. Conservation of a specificalternative splicing pattern throughout evolutionary history providesstrong evidence of biological function (Mironov et al., 1999).Herein, alternative AQP2 is likely functional and it must have beenacquired quite early in the evolution of modern cetaceans and highlyconserved during evolution.

Immunohistochemistry revealed different distribution patterns ofAQP2 and alternative AQP2. In the kidney, AQP2 was observed atthe apical membrane of the collecting duct, whereas alternativeAQP2 was observed in a variety of structures. Alternative AQP2protein was also detected in many organs (Fig. S1) and these dataare coincident with the results of RT-PCR, suggesting thatalternative AQP2 may be ubiquitously localized in the dolphinbody. In the renal collecting duct, alternative AQP2 was found inboth the cytoplasm and the plasma membrane, while AQP2 wasrestricted to the plasma membrane, as observed in forced expressionof AQP2 isoforms in Xenopus oocytes. This difference in isoformdistribution may be related to differences in their amino acidsequences. The deduced amino acid sequence of alternative AQP2lacks phosphorylation sites at the C terminus of AQP2 responsiblefor trafficking to the (apical) membrane (Hoffert et al., 2006). Thelack of these sites in dolphin alternative AQP2 may impair thetrafficking efficiency of the protein to the plasma membrane,leading to lower water permeability and freedom from the control ofvasopressin. These data suggest that alternative AQP2 had lowerlevels of water flux through plasma membrane than normal AQP2and it would be less effective at osmotic water regulation on theplasma membrane as well as on the membrane of organelles.Further investigations, especially on the details of the intracellulardistribution, are necessary to unveil the specific function ofalternative AQP2.

Expression of both AQP2 isoforms was induced by hypertonicitydue to additional NaCl. The renal medullary cells are constantlybathed in a hypertonic interstitial solution of varying concentrationsthat is required for concentrating urine. AQP2 is a molecule thatresponds to this hypertonic environment (Kwon et al., 2009). Thetotal amount of AQP2 in epithelial cells of the renal collecting duct istightly regulated by hormones such as vasopressin. AQP2 expressionis also induced by hypertonicity (Hasler, 2009; Kasono et al., 2005;Storm et al., 2003), and this response may occur via TonEBP, atranscription factor that may play an osmosensitive role in cellregulation during hypertonic stress (Miyakawa et al., 1999). Underhypertonic conditions, TonEBP is expressed and binds to thetonicity-responsive enhancer cis element upstream of AQP2 toinitiate its expression (Miyakawa et al., 1999). TonEBP also

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Fig. 2. Water permeability of Xenopus laevis oocytes injected with cRNAof AQP2 or alternative AQP2 from the bottlenose dolphin. (A) Osmoticoocyte volume (V ) change in oocytes injected with water (control), dolphinAQP2 cRNA, dolphin alternative AQP2 or 0.03 mmol l−1 l−1–HgCl2 (+Hg).(B) Water permeability of oocytes (mean±s.e.m.) in each treatment group.Values marked with different letters are significantly different from each other(ab, ac, bd, be, ce: P<0.01; ad, bc, cd, de: P<0.05). (C) Fluorescentimmunostaining of oocytes injected with cDNA of AQP2 (i) or alternative AQP2(ii) and control (iii).

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participates in compatible osmolyte accumulation by upregulatingrelevant genes (Coleman et al., 2000; Miyakawa et al., 1998; Rimet al., 1998). The DNA sequence of the TonEBP binding site in theAQP2 promoter is GGAAA (Halterman et al., 2012); the bottlenosedolphinAQP2 proximal promoter includes this sequence at−139 and

−327 bp upstream of the start codon (turTru1: GeneScaffold_1570,Ensembl Genome Blowser). The TonEBP gene is present in thebottlenose dolphin (ENSTTRG00000010017), and we confirmedthat TonEBP mRNA is expressed and upregulated by addition ofNaCl in dolphin renal cells. Collectively, these observations indicate

Alternative AQP2

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Absorbed-anti-alternative AQP2

1918171615141312111098765432Fig. 3. Expression of mRNA for AQP2 and alternativeAQP2 in the bottlenose dolphin. (A) Semi-quantitativeRT-PCR for AQP2 and alternative AQP2 with cDNAs from19 organs. Numbers represent organs as follows: 1, brain;2, pituitary; 3, epidermis; 4, muscle; 5, heart; 6, kidney; 7,adrenal gland; 8, testis; 9, ovary; 10, placenta; 11, forestomach; 12, main stomach; 13, pyloric stomach; 14, smallintestine; 15, large intestine; 16, liver; 17, pancreas; 18,spleen; and 19, lung. Lengths of the RT-PCR products are913, 813 and 228 bp for AQP2, alternative AQP2 andGAPDH, respectively. (B) Immunohistochemistry for AQP2and alternative AQP2 in the renal cortex, outer medulla andpapilla, and immunoblotting for the aquaporins in the renalmedulla. Rabbit IgG was used instead of antibodies for anegative control. To be certain of the specificity of theantibody for alternative AQP2, an antibody absorptiontreatment was carried out, using peptide antigen. C, renalcalix; CD, collecting duct; E, transitional epithelium; M,molecular marker.

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that transcription of the dolphin AQP2 isoforms is induced byhypertonicity due to increased NaCl via TonEBP, as in othermammals. In addition, the fact that dolphin renal cells in controlgroup survived in medium with increased NaCl levels but cells withsiRNA knockdown for both of the dolphin AQP2 isoforms could notsurvive suggest that AQP2 and alternative AQP2 may play roles incellular osmotic regulation.The high degree of alternative AQP2 sequence conservation

among members of the order Cetacea, its ubiquitous expression indifferent body tissues and its hypertonicity-responsive expressionsuggest that this alternatively spliced protein plays a role in cellularosmoregulation throughout the body of this group of aquatic

mammals living in hypertonic environments. Several studies haverecently reported that AQP2 was positively selected for during thesecondary adaptation in cetacean species, suggesting that theevolution of the gene was an important event in the development ofenhanced capacity for water reabsorption in the renal tubules (Neryet al., 2013; Xu et al., 2013; Yim et al., 2014). Extensive informationcan be obtained from genome-scale analyses, providing insight intothe nature and extent of selective pressures that contributed to theevolution of a particular group. However, the physiological functionsof each molecule, especially on unanticipated products such asalternatively spliced proteins, could be overlooked by genomicanalyses in certain instances. Our findings reaffirm that the inferenceson physiological mechanisms and the functional relevance of each

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Fig. 4. Effects of medium with additional NaCl or mannitol on the mRNAexpression of AQP2 isoforms in cultured renal cells of bottlenosedolphin. Expression of (A) AQP2 mRNA and (B) alternative AQP2 mRNA.NaCl or mannitol was added to the culture to increase the extracellularosmolality to 350 or 500 mOsm kg−1. Control used normal medium(255 mOsm kg−1). Values are presented as % change in mRNA quantity fromthe control. (C) Amplification of the full-length ORF of TonEBP using cDNAfrom cultured renal cells and effects of NaCl addition on its mRNA expression.Values marked with different letters are significantly different from each other(ab, df, ef: P<0.01; bc, de: P<0.05). M, molecular marker.

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Fig. 5. Effects of siRNA knockdown of AQP2 and alternative AQP2 incultured bottlenose dolphin renal cells. (A) Viability of cells (control, siRNAfree, universal negative control siRNA, AQP2 siRNA or alternative AQP2siRNA) after NaCl addition as assayed by cellular ATP contents. Values arepresented as % change in ATP contents (mean±s.e.m.) from that of controls.siRNA free indicates cells treated with Lipofectamine 3000 without siRNA.(B) Relative luciferase activity is shown in cells transfected with GAPDH, AQP2or alternative AQP2 siRNA. Values are presented as % change (mean±s.e.m.)in luminescence intensity from that of controls. Values marked with differentletters and asterisks are differed significantly from each other (ab, ac:P<0.0001; bc, **: P<0.01).

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molecule should be confirmed by investigative approaches based onfunctional studies.

CONCLUSIONSThis study revealed that (1) two alternatively spliced isoforms ofaquaporin 2 (AQP2 and alternative AQP2) are expressed in thebottlenose dolphin, (2) both isoforms are upregulated byhypertonicitydue to increased NaCl, (3) knockdown of the isoforms resulted in celldeath in high NaCl hypertonic medium, (4) alternative AQP2 isexpressed in every organ examined, in contrast to normal AQP2,which is expressed only in the kidney, and (5) the alternative splicingmost probably occurs only in cetacean species. These results suggestthat cetaceans may have utilized the abilities of AQP2, which isresponsive to hypertonicity and functions in the kidney (in whichintra- and extracellular concentrations of urea and NaCl fluctuateduring antidiuresis and diuresis), in the whole body as an antidote tothe hypertonic external medium when they radiated back into the

marine environment. Further detailed studies on the intracellularfunction of alternative AQP2 will offer interesting suggestions on thephysiological adaptation of cetaceans to aquatic life.

AcknowledgementsWe sincerely thank A. Takayama and S. Moriya (Nihon University) for theircooperation in experiments. We also appreciate K. Noguchi (Yamaguchi University,Yamaguchi, Japan) for his great contribution to establishing the Dol KT1 cell line.We thank Taiji Fisheries Cooperative Union, Wakayama, Japan, Dr T. Iwasakiand T. Hara (Fisheries Research Agency, Kanagawa, Japan), K. Tokutake andDr K. Ueda (Okinawa Churaumi Aquarium, Okinawa, Japan), K. Okutsu (YokohamaHakkeijima Sea Paradise, Kanagawa, Japan), Dr T. Minakawa (Ibaraki PrefecturalOarai Aquarium, Ibaraki, Japan) and Dr K. Kohyama (Izu Mito Sea Paradise,Shizuoka, Japan) for their cooperation in providing specimens.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsM.S. conceived the study, conducted the field work, interpreted results, and drafted,revised and edited the manuscript. M.S., H.W., T.I., T.S., Y.I., K.Y. and K.K. processedsamples and analyzed data. All authors approved the final version of the manuscript.

FundingThis study was funded by a Grant-in-aid for Scientists from the Japan Society for thePromotion of Science (C 23580265, C 26450292) and a Nihon University IndividualResearch Grant (2010 and 2013) to M.S., and by International Joint Research andTraining of Young Researchers for Zoonosis Control in the GlobalizedWorld to M.S.,T.I. and T.S. The funders had no role in study design, data collection and analysis,decision to publish, or preparation of the manuscript.

Data availabilityDNA Data Bank of Japan accession numbers for AQP2 and alternative AQP2 inbottlenose dolphin are LC053641 (http://getentry.ddbj.nig.ac.jp/getentry/na/LC053641) and LC053642 (http://getentry.ddbj.nig.ac.jp/getentry/na/LC053642),respectively.

Supplementary informationSupplementary information available online athttp://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.132811/-/DC1

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B. B., Petersen, P., Rinvik, E., Torgner, I. A. and Ottersen, O. P. (2005). Brainmitochondria contain aquaporin water channels: evidence for the expression of ashort AQP9 isoform in the innermitochondrialmembrane.FASEBJ.19, 1459-1467.

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Tursiops

Lagenorhynchus

Pseudorca

Neophocaena

Kogia

Berardius

Balaenoptera

Bos

Bison

Ovis

Pantholops

Sus

Camelus10 MYA

Exon 3 Intron 3

Newly acquired exonin Cetacea

Exon 4

Tursiops

Lagenorhynchus

Pseudorca

Neophocaena

Kogia

Berardius

Balaenoptera

Bos

Bison

Ovis

Pantholops

Sus

Camelus

A

BC

etac

eaR

umin

antia

A

rtiod

acty

laA

rtiod

acty

laC

etac

ea

Fig. 6. Alignment of the region through the third intron of AQP2 ofcetacean and artiodactyl species usingmVISTA. (A) Alignments of Cetacea(upper) and Ruminantia (lower) with the bottlenose dolphin (Tursiops)sequence and that of cow (Bos) as a reference, respectively, are shown with aphylogenic tree. (B) Alignment among cetacean and artiodactyl species withthe sequence in bottlenose dolphin as a reference. The blue, light blue andpink columns in AQP2 of the bottlenose dolphin/cow indicate authentic exons,the newly acquired exon in Cetacea, and the intron, respectively. The height ofthe shading represents the percent identity between two sequences. Theareas between the two dashed lines in A indicate the corresponding region inAQP2 of other mammals.

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